(1) Field of the Invention
The present invention relates to a method for inhibiting blood procoagulant activity of a histocompatibility antigen ((HLA). In particular, the present invention relates to the use of an enterotoxin to inhibit the activity, particularly in preventing graft rejection coagulation and malignancy induced coagulation.
(2) Prior Art
In 1865, Trousseau observed a high incidence of venous thrombosis in patients with gastric carcinoma and described the syndrome which bears his name (Trousseau, A., In Clinique Medicale de l'Hotel-Dieu de Paris; Paris, Balliere, 3:654 (1865)). A considerable body of evidence supports an association between cancer and thromboembolic disorders (Rickles, F. R., et al., Blood 62:14-31 (1983) ) and elements of the hemostatic system (i.e. platelets, thrombin and fibrin) are proposed as causal for tumor cell metastasis (Weiss, L., et al., Clin. Expl. Metas. 7:127-167 (1989)). Activated platelets may enhance tumor cell adhesion to the vessel wall (Honn, K. V., et al., Biochem. Pharm. 34:235-241 (1985)) and induce endothelial cell retraction (Honn, K. V., et al., FASEB. J. 3:2285-2293 (1989)) while fibrin may aid tumor cells to escape the cellular immune system (Gorelik, E., Cancer Res. 47:809-815 (1987)). Tumor cell activation of platelets and generation of fibrin may require thrombin. Therefore, the identification and isolation of the tumor cell prothrombogenic protein(s) responsible for thrombin generation are of considerable interest.
At least four separate factors are thought to mediate tumor cell induced thrombin generation. The first is tissue factor, a transmembrane glycoprotein and receptor, also associated with normal tissue (Broze, G. J., et al., J. Biol. Chem. 260:10917-10920 (1985); and Buha, A., et al., Proc. Natl. Acad. Sci. USA 83:299-302 (1986)). The second, cancer procoagulant, is a cysteine proteinase that directly activates factor X (Gordon, S. G., et al., J. Clin. Invest. 67:1665-1671 (1981)). The third is a protein termed platelet aggregating activity/procoagulant activity (PAA/PCA) isolated from solid tumors or from tumor cells grown in culture which lacks proteolytic activity and is dependent upon factor X for activity (Cavanaugh, P. G., et al., Thromb. Res. 37:309-326 (1985)). More recently, a factor Xa receptor has been identified on some tumor cells (Sakai, T., et al., J. Biol. Chem. 265:9105-9113 (1990)).
Anticoagulant therapy with warfarin and other coumarins consistently results in a reduction of lung colony formation and spontaneous metastasis strongly suggesting that components of blood coagulation facilitate metastasis. However, warfarin may produce direct effects on tumor cells independent of its action as an anticoagulant (Donati, M. B., et al., In: Recent Advances in Blood Coagulation (Ed. L. Poller), Churchill Livingstone, Edinburgh, pp. 227-259 (1982)). O'Meara and his associates were the first to study a tumor-derived procoagulant activity (O'Meara, R. A. Q., Irish J. Med. Sci. 394:474-479 (1958)). Four separate procoagulants have thus far been identified and are described below.
Tissue factor--Kinjo et al (Kinjo, M., et al., J. Cancer 39:15-23 (1979)), Hudij and Bajaj (Hudig, D., et al., Thromb. Res. 27:321-332 (1982)), Khato, et al. (Khato, J., et al., GANN 65:289-294 (1974)), and Dvorak, et al. , (Dvorak, H. F., et al., Cancer Res. 43:4334-4342 (1983)) have shown that tumor cell induced coagulation required FVII suggesting that thromboplastin was responsible for activity. This has been substantiated by showing that antibodies to human brain thromboplastin neutralize the procoagulant activity of human leukemic promyelocytes (Gouault-Heilmann, M., et al., J. Haematol. 30:151-157 (1975)). Tissue factor has been purified only from normal brain tissue by affinity chromatography (Broze, G. J., et al., J. Biol. Chem. 260:10917-10920 (1985); and Guha, A., et al., Proc. Natl. Acad. Sci. USA 83:299-302 (1986)). It has a molecular weight of 43,000, requires lipids for activity, and catalyses the hydrolysis of factor IX and X by factor VIIa.
Cancer procoagulant--Gordon and co-workers (Gordon, S. G., et al., Cancer Res. 38:2467-2472 (1978); Gordon, S. G., et al., J. Clin. Invest. 67:1665-1671 (1981); Gordon, S. G., et al. , Thromb. Res. 6:127-137 (1975); Gordon, S. G., J. Histochem. Cytochem. 29:457-463 (1981); Gordon, S. G., et al., J. Natl. Cancer. Inst. 62:773-776 (1979); and Falanga, A., et al., Biochemistry 24:5558-5567 (1985)) purified cancer procoagulant (CP) from a soluble extract of rabbit V2 carcinoma cells. Purified CP has a molecular weight of 68,000 with an isoelectric point of 4.8. CP(i) requires phospholipid for activity (ii) activates factor X and this proteolytic activity can be inhibited with iodoacetamide and mercury and reactivated with potassium cyanide, dithiothreitol and EDTA, suggesting that it may be a cysteine proteinase and (iii) is stable in alkaline pH (Barrett, A. J., et al., Mammalian Proteases Vol. 1 Endopeptidases 416 pp. Academic Press, New York (1980)); thus, it apears to be a non-lysosomal cysteine proteinase.
Platelet aggregating activity/procoagulant activity (PAA/PCA)--Cavanaugh et al have shown that yet another procoagulant protein PAA/PCA is produced by tumor cells. The size of this protein varies with species, the murine adenocarcinoma protein has a molecular weight of 51,000 (Cavanaugh, P. G., et al., Thromb. Res. 37:309-326 (1985)) while the rat Walker 256 carcinosarcoma produces a protein of 58,000 daltons (Cavanaugh, P. G., et al., Hemostasis, 18:37-46 (1988)). PAA/PCA (i) does not have any proteolytic activity and (ii) procoagulant activity is observed only after reconstitution with phospholipids.
Factor Xa receptor on tumor cells--Recent studies by Sakai and Kisiel (Sakai, T., et al., J. Biol. Chem. 265:9105-9113 (1990)) and Altieri and Edgington (Altieri, D. C., et al., J. Biol. Chem. 264:2969-2977 (1989)) provide evidence for the presence of a novel prothrombinase complex in some tumor cells. Radio-iodinated FXa binds specifically to a human hepatocellular carcinoma cell line (HepG2) which constitutively synthesizes a factor V/Va molecule and to a human bladder carcinoma (J82) which does not synthesize factor V/Va. Pretreatment of J82 cells with anti-human V IgG had no significant effect on binding of FXa or prothrombin activation. However, pretreatment of HepG2 cells with FV antibodies inhibited prothrombin activation in a dose dependent manner without inhibiting FXa binding. When both cells were incubated with exogenous FVa, the binding of FXa to either HepG2 or J82 cells were only marginally affected. These studies suggest FXa binding to sites proximal to but independent of cell surface FVa.
Thus, procoagulant activities associated with tumor cells or attached stromal cells would promote the generation of thrombin. Thrombin is a potent and physiologic activator of platelets and it would be intuitive that platelets would be activated in the vicinity of tumor cells. Indeed, intravenously injected tumor cells were observed to induce platelet aggregation (Gastpar, H., J. Med. 8:103-114 (1977)). Thrombocytopenia induced by neuraminidase or antiplatelet antiserum results in decreased lung colony formation from tail vein-injected tumor cells (Gasic, G. J., et al., Proc. Natl. Acad. Sci. USA 61:46-52 (1968); Gasic, G. J., et al., Int. J. Cancer 11:704-718 (1973); Pearlstein, E., et al. , Cancer Res. 44:3884-3887 (1984); Skolnick, G., et al., J. Cancer Res. Clin. Oncol. 105:30-37 (1983); Ivarrson, L., In:Acta Chirurgica Scandinavia (Eds. Almqvist and Wiskell), Stockholm, (1976); and Sindelar, W. R., et al., J. Surg. Res. 18:137-161 (1975)) and spontaneous metastasis from subcutaneous tumors (Pearlstein, E., et al., Cancer Res. 44:3884-3887 (1984); and Wood, S., Jr., et al., Bull. Schweiz. Akad. Med. Wiss. 20:92-121 (1964)) suggesting a role for platelets in metastasis. Fibrin adhered to tumor cells may also play a role in tumor growth and invasion (Gastpar, H., J. Med. 8:103-114 (1977)).
Numerous examples abound in the literature providing strong evidence that there is a definite association between the incidence of thromboembolic complications and onset of tissue transplant rejection. Foegh and coworkers (Foegh, M. L., et al., Urine i-TXB2 in renal allograft rejection, Lancet. 2:431-434 (1981)) first observed an increase of thromboxane B2 preceeding the onset of renal graft rejection. There are now several papers on the association of thromboembolic disorders with liver (Blumhardt, G., et al., Transplantation Proc. 19:2219-2220 (1987); and Hidalgo, E. G., et al., Hepato-Gastroenterology. 36:529-532 (1989)), pancreatic (Boiskin, I., et al., Am. J. Roentgenology. 154:529-531 (1990); and Soon-Shiong, P., et al., Transplant Proc. 20:965-971 (1988)) and bone marrow (Rio, B., et al., Blood. 67:1773-1776 (1986)) graft rejection.
Some success has been achieved in engraftment of renal transplants by treatment of recipients with cyclosporin A (CsA). This immunosuppressive regimen however does not overcome the small number of allografts lost in early post-transplantation as a result of renal artery and vein thrombosis (Arrunda, J. A. L., et al., Renal vein thrombosis in kidney allografts. Lancet. 2:585 (1973)). There have also been reports of an increased incidence of thrombosis in patients immunosuppressed with CsA (Merrion, R. M., et al., Transplant Proc. 17:1746-1750 (1985)). An added complication is nephrotoxicity (Myers, B. D., et al., N. Engl. J. Med. 311:699-705 (1984); and Ryffel, B., et al., Toxicol Pathol 14:78 (1986)) and hepatotoxicity (Klintmalm, G. B., et al., Transplantation 32: 488-489 (1981); and Roger, S., et al., Transplant Proc. 15:2761-2767 (1983)) caused by high doses of CsA.
More recently, two additional drugs have become available for the treatment of graft rejection. FK506, an antibiotic of the macrolide family isolated from Streptomyces tsukubaensis (Kino, T., et al., J. Antibiotic 40:1256-1265 (1987)) is about 100 to 500 times more potent than CsA. Rapamycin, which was recently shown to have immunosuppressive effects (Martel, R. R., et al., J. Physiol. Pharmacol. 55:48-(1977)) was originally identified for its antifungal activity (Dumont, F. J., et al., J. Immunol. 144:251-258 (1990)), is a structural analog of FK506 isolated from Streptomyces hygroscopicus (Sehgal, S. N., et al., J. Antibiot. 28:727-(1979)).
All three immunosuppressants exert their effects by binding to proteins termed immunophilins with high affinity. CsA binds to human cyclophilin (Handsschumacher, R. D., et al., Science, 226:544-547 (1984)) and both FK506 and rapamycin bind to human FK506 binding protein (FKBP) (Harding, M. W., et al., Nature, 341:758-760 (1989)). It has been suggested that FK506 like CsA inhibits a T cell receptor mediated signal transduction pathway that results in the transcription of early T cell activation genes, including interleukin-2 (IL-2). (e). Although rapamycin binds to FKBP, it has no effect on IL-2 production. In fact it appears to behave antogonistically to FK506 (Dumont, F. J., et al., J. Immunol. 144:1418-1424 (1990)). Furthermore, drug combinations of FK506 and CsA, FK506 and rapamycin and rapamycin and CsA are not synergistic (Metcalf, S. M., et al., Transplantation. 49:798-802 (1990)). Recent studies have also shown that FK506 may have diabetogenic effects (Calne, R., et al., Transplant Proc. 19 (suppl 6): 63 (1987)). It would appear that these immunosuppressants would have no influence on thrombotic consequences imposed by tissue transplants. Indeed one clinical study showed that administration of subcutaneous heparin together with a regimen of CsA significantly reduced thromboembolic complications and prolonged graft survival (Ubhi, C. S., et al., Transplantation 48:886-887 (1989)).
It has been thought that other factors may mediate coagulation in graft rejection and tumor cell induced coagulation. To date, such other factors and a method of inhibiting the factor has been unknown.